PROJECT SUMMARY Radiation therapy is standard management for glioblastoma (GBM); however, these tumors invariably recur, frequently in a more aggressive and treatment-resistant manner than the original tumor. Radiation is known to induce alterations correlated with mesenchymal tumor subtype. However, the mechanisms by which the radiated microenvironment may exacerbate recurrent tumor behavior remain Ill-defined. Cell senescence is induced by radiation and given pro-inflammatory and ECM-altering factors secreted by senescent cells, may contribute to worsened GBM behavior in the previously radiated brain. ? Our long-term goal is to understand the contribution of the radiated tumor microenvironment, including that of senescent cells to facilitate tumor recurrence. ? The objective of these proposed activities is to evaluate the generalizability and reproducibility of radiation- induced pro-tumorigenic impacts across multiple parameters of radiation dose, cell type, and model, and to interrogate the role of senescent cells in this process. ? Our central hypothesis is that brain radiation alters the brain microenvironment, including via induction of cellular senescence, to increase tumor aggressiveness. ? The specific aims of this proposal are: (Aim 1) to rigorously test if prior radiation exposure induces more aggressive tumor behavior across radiation schemas, GBM models and post-radiation time-points; (Aim 2) to test if senescent cells in the glioblastoma microenvironment increase tumor aggressiveness, using gain and loss of function experiments with senescent cell implantation and pharmacologic and genetic ablation of senescent cells. As an experimental sub-aim, we will test using Single nucleus-RNAseq if transcriptional changes of senescence are induced in a specific stromal cell types, and whether or not these correlate with induction of a mesenchymal signature in implanted tumor cells. With advances in senolytic therapies, this work may provide an avenue to attenuate unintended deleterious sequelae of radiation therapy on the tumor microenvironment. Moreover, given the de-novo emergence of senescence in GBM, senolytic therapies may provide an avenue to attenuate GBM progression both in synergy with, and independently of brain radiation.